Chemical reactions in biological systems are almost always facilitated by the action or one or more catalysts. Enzymes, which are proteins that catalyze biological reactions, are known for their catalytic efficiency and specificity. Enzymes typically accelerate reactions by factors of 1 million or more. Many reactions in biological systems do not occur at perceptible rates in the absence of enzymes.
Enzymes are highly specific in the type of reaction catalyzed as well as in the particular substrates which are acted upon. One broad category of enzymes includes the proteolytic enzymes which catalyze the hydrolysis of peptide bonds. Proteolytic enzymes, also known as proteases, vary significantly in their degree of specificity. For example, subtilisin, which comes from certain bacteria, will cleave peptide bonds regardless of the nature of the side chains adjacent to the bond. Trypsin is quite specific in that it splits peptide bonds on the carboxyl side of lysine and arginine residues only. Thrombin, an enzyme participating in blood clotting, is even more specific than trypsin. Thrombin only cleaves between arginine and glycine residues. These are only a very few examples of proteases; many other proteases are known. There are several general categories of protease. These categories include serine, crysteine, aspartic, and metalloproteases. This classification is based on the most prominent functional group at the active site of the proteases. The serine proteases are of particular interest relative to the current invention.
Much information exists on the molecular structure and function of many serine proteases from diverse species. The majority of these enzymes consist of a single polypeptide chain of molecular weight 25,000-30,000. Chymotrypsin and subtilisin are both members of the serine protease family. Like other proteases, serine proteases cleave peptide bonds within a polypeptide to produce two smaller peptides. The cleavage reaction will typically proceed through an intermediate transition state which is facilitated by the presence of the protease. For serine proteases, the formation of an acyl-enzyme intermediate involving a reactive serine residue is the first step in the hydrolysis reaction. Deacylation of the acyl-enzyme intermediate is the second step in the hydrolysis. Like other proteases, serine proteases achieve their catalytic activity by lowing the activation energy for a specific hydrolysis reaction.
Proteases can be obtained from a wide variety of sources including fungi, bacteria, and eukaryotic cells. Although proteases have been obtained from many bacteria, relatively few proteases have been identified from bacteria which are known to live in extremely hot environments. Bacteria capable of growing at or above 80.degree. C.-100.degree. C. are generally known as extreme thermophiles or hyperthermophiles. Such highly thermophilic microorganisms have been the object of considerable scrutiny by researchers attempting to gain insight into the biochemical mechanism which enables these microbes to survive under such extreme conditions.
A number of microorganisms have been isolated from extremely hot environments. These microorganisms have been studied and certain useful compounds have been identified. For example, thermostable DNA polymerase have been obtained from Thermus aquaticus. Proteases have been isolated from thermophiles including T. aquaticus, Desulfurococcus species, Pyrococcus furiosus, Sulfolobus acidocaldarius, Thermococcus stetteri, and Pyrobaculum aerophilum. However, difficulties in culturing extremeophiles have limited the number of these microbes which have been characterized as well as the number of useful compounds isolated therefrom (Brennan, Chemical and Engineering New, Oct. 14, 1996).
Stetter, et al. identified microorganisms from the hot springs of Vulcano Island, Italy, that flourish at temperatures exceeding 100.degree. C. (Stetter, K. O. "Microbial Life in Hyperthermal Environments," ASM News 61:285-290, 1995; Stetter, K. O., Fiala, G., Huber, R. And Segerer, A. "Hyperthermophilic Microorganisms," FEMS Microbiol. Rev. 75:117-124, 1990). While thermophilic organisms that grow optimally at 60.degree. C. have been known for many years, the hyperthermophilic (or extremely thermophilic) microorganisms belong to a new evolutionary class called Archaea (Woese, C. R., Kandler, O. and Wheelis, M. L. "Towards a Natural System of Organisms: Proposal for the Domains Archaea, Bacteria, and Eucarya," Proc. Natl. Acad. Sci. USA 87:4576-4579, 1990). The Archaea are believed to have originated over a billion years ago during the epoch when the Earth was cooling. Consequently their evolutionary development was set in motion within the environment of hot springs and deep sea hydrothermal vents. One member of this new group is Pyrococcus furiosus which grows optimally at 100.degree. C.-110.degree. C. (Fiala, G. and Stetter, K. O. "Pyrococcus furiosus s. Nev. Represents a Novel Genus of Marine Heterotrophic Archaebacteria Growing Optimally at 100.degree. C.," Arch. Microbiol. 145:56-61, 1986). Pyrococcus furiosus is an obligate heterotroph that can be grown on polymeric substrates including protein and starch at temperatures of up to about 103.degree. C. Preparations containing proteolytic enzymes prepared from Pyrococcus furiosus have been previously described in U.S. Pat. Nos. 5,242,817 and 5,391,489. These patents do not describe the enzymes identified by the current applicant. Other publications describing proteases from P. furiosus also do not describe the current enzymes. See, for example, Blumentals, Ilse I., Robinson, Anne S., and Kelly, Robert M., "Characterization of Sodium Dodecyl Sulfate-Resistant Proteolytic Activity in the Hyperthermophilic Archaebacterium Pyrocuccus furiosus." Applied and Environmental Microbiology, 56,7:1992-1998, (1990); Eggen, Rik, Geerling, Ans, Watts, Jennifer and de Vos, Willem M., "Characterization of pyrolysin, a hyperthermoactive serine protease from the archaebacterium Pyrococcus furiosus." FEMS Microbiology Letters, 71:17-20 (1990); Voorhorst, Wilfried G. B., Eggen, Rik I. L., Geerling, Ans C. M., Platteeuw, Christ, Siezen, Roland J., de Vos, Willem M., "Isolation and Characterization of the Hyperthermostable Serine Protease, Pyrolysin, and Its Gene from the Hyperthermophilic Archaeon Pyrococcus furiosus." Journal of Biological Chemistry, 271,34:20426-20431 (1996).
The use of proteolytic enzymes for selective peptide bond synthesis has been previously investigated. The majority of studies so far on protease-mediated peptide synthesis have utilized what has been called "semi-synthesis". In these reactions, the acyl donor is a substrate for the enzyme (amide or ester). The substrate is utilized to acylate the enzyme (e.g., a serine or thiol protease) followed by deacylation by C-terminally blocked amino acid or peptide. (See Nakatsuka, T., Sasaki, T., and Kaiser E. T. "Peptide Segment Coupling Catalyzed by the Semisynthetic Enzyme Thiolsubtilisin." J. Am. Chem Soc. 109:3808-3810, 1087; Abrahmsen, L., Tom, J., Burnier, J., Butsher, K. A., Kossiakoff, A., and Wells, J. A., "Engineering Subtilisin and its Substrates for Efficient Ligation of Peptide Bonds in Aqueous Solution." Biochemistry 30:4151-4159, 1991; Christenen, U., Drohse, H. B., and Molgaard, L., "Mechanism of Carboxypeptidase-T-catalyzed Peptide Semisynthesis" Eur. J. Biochem., 210:467-473, 1992.
The ability to synthesize peptides and ligate polypeptides in aqueous solution under controlled conditions would be highly advantageous. Current protein synthesis methodologies result in much reactant and solvent toxic waste, which must be disposed of.